DNA tetrahedral nucleic acid framework type gastric cancer diagnosis-treatment integrated reagent and preparation method and application thereof

A tetrahedral and framework-type technology, which can be used in preparations for in vivo experiments, gene therapy, pharmaceutical formulations, etc., and can solve the problem of a small number of targeted nanoprobes.

Active Publication Date: 2020-10-23
NANJING UNIV OF POSTS & TELECOMM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Purpose of the invention: In order to overcome the defect that the number of targeted nanoprobes for integrated diagnosis and treatment is relatively small, the present invention provides a drug-resistant gastric cancer cell-targeted gastric cancer micronucleic acid marker miRNA imaging and chemotherapy and gene dual A DNA tetrahedral nucleic acid framework type gastric cancer diagnosis and treatment integrated reagent for mode synergistic treatment and a preparation method thereof. The integrated reagent, that is, a nano-probe uses a DNA tetrahedral nucleic acid framework as a main body, loads a commonly used chemotherapeutic drug DOX, and then further builds on the DNA tetrahedron. Linking the MUC1 aptamer that can target drug-resistant gastric cancer cells and the Mcl-1 siRNA that has the effect of silencing Mcl-1 protein, a novel drug that can be used for tumor cell target recognition, binary miRNA imaging, and chemotherapy and gene dual Multifunctional nanoagents for modality synergistic therapy

Method used

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  • DNA tetrahedral nucleic acid framework type gastric cancer diagnosis-treatment integrated reagent and preparation method and application thereof
  • DNA tetrahedral nucleic acid framework type gastric cancer diagnosis-treatment integrated reagent and preparation method and application thereof
  • DNA tetrahedral nucleic acid framework type gastric cancer diagnosis-treatment integrated reagent and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Synthesis of DNA tetrahedral nucleic acid framework

[0062] This design synthesizes the DNA tetrahedral nucleic acid framework through a simple one-step synthesis method, and the synthesis effect will be characterized and analyzed by PAGE gel electrophoresis.

[0063] 1) Each single strand of ssDNA A, ssDNA B, ssDNA C, and ssDNA D was diluted to 100 μM with ultrapure water.

[0064] 2) The above samples were prepared and mixed together, vortexed and diluted to 3 μM with 2×PBS, incubated at 95°C for 10 minutes, and cooled at 37°C for 1 hour to obtain the DNA tetrahedral nucleic acid framework.

[0065] 2. Construction of integrated reagent for diagnosis and treatment of gastric cancer based on DNA tetrahedral nucleic acid framework

[0066] 1) Add the DOX solution to the prepared DNA tetrahedral nucleic acid framework, react at 37°C for 3h, add the product to an ultrafiltration centrifuge tube, centrifuge at 6000rpm for 10min, add water and then centrifuge twice to ...

Embodiment 2

[0068] Example 2 DNA Tetrahedral Nucleic Acid Framework Assembly Process and Reaction Mechanism Characterization

[0069] The working principle of the DNA tetrahedral nucleic acid framework is as follows figure 1 As shown, in order to verify the correctness of the mechanism, 10% PAGE gel was used for gel electrophoresis experiments. 5 μL of DNA samples were mixed with 1 μL of 6×DNA loading buffer, and imaged after running at 80V for 90 minutes.

[0070] The DNA samples correspond to the DNA samples in different swimming lanes, namely: swimming lane 1: ssDNA A; swimming lane 2: ssDNA B; swimming lane 3: ssDNA C; swimming lane 4: ssDNA D; swimming lane 5: ssDNA A and ssDNA B equal hybridization; lane 6: ssDNA A and ssDNA C equal hybridization; lane 7: ssDNA A and ssDNA D equal hybridization; lane 8: ssDNA B and ssDNA C equal hybridization; lane 9: ssDNA B and ssDNA D Equal hybridization; Lane 10: Equal hybridization of ssDNA C and ssDNAD; Swimming lane 11: Equal hybridization o...

Embodiment 3

[0076] Example 3 Detection of miRNA by DNA tetrahedral nucleic acid framework-type integrated reagent for diagnosis and treatment of gastric cancer

[0077] To the DNA tetrahedral nucleic acid framework type gastric cancer diagnosis and treatment integrated reagent constructed in Example 1, add miRNA-106a or miRNA-106a or miRNA- 21. The final concentration of the reagent is 50nM. After incubating at 37°C for two hours, use a fluorescence spectrometer to test its fluorescence intensity.

[0078] like figure 1 As shown, when miRNA-106a exists, miRNA-106a will be replaced by the DNA strand with Cy3 dye on one edge of the DNA tetrahedral nucleic acid framework reagent, so that Cy3 and the quencher BHQ-2 that were originally close to each other are far away from each other Cause Cy3 signal recovery; similarly, when miRNA-21 exists, miRNA-21 will be replaced by the DNA strand with Cy5 dye on the other edge of the DNA tetrahedral nucleic acid framework reagent, so that Cy5 and quenc...

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Abstract

The invention discloses a DNA tetrahedral nucleic acid framework type gastric cancer diagnosis-treatment integrated reagent which includes a DNA tetrahedral nucleic acid framework which is loaded withchemotherapeutic drugs, and is obtained by connecting a MUC1 targeting aptamer with Mcl-1 siRNA. The invention further discloses a preparation method and application of the integrated reagent. The integrated reagent can perform targeting recognition of drug-resistant gastric cancer cells SGC-7901/ADR, miRNA detection imaging and chemical drugs and gene silencing combined synergistic treatment, the internalization efficiency of the reagent in the drug-resistant gastric cancer cells and the detection efficiency of miRNA are improved, and by using the structural changes of the nucleic acid framework in a miRNA detection process, the rapid release of DOX loaded in a DNA double strand is realized. The reagent is further combined with Mcl-1 siRNA, the gene silencing of the Mcl-1 siRNA significantly improves the therapeutic effect of DOX, and the drug resistance problem in the application of nanomedicine in biological systems is solved.

Description

technical field [0001] The invention belongs to the technical field of functional nano-probes, and in particular relates to a DNA tetrahedron nucleic acid framework-type gastric cancer diagnosis and treatment integrated reagent and its preparation method and application. Background technique [0002] DNA Tetrahedral Nucleic Acid Framework (DNA Tetrahedral Nucleic Acid Framework), as one of the simplest and strongest pyramid-shaped three-dimensional structure models, has high mechanical rigidity and stability, rich functional modification sites, and simple production process , with high yield, showing good application prospects in in vivo imaging, gene carrier, drug delivery, biosensing detection, etc. The DNA tetrahedral nucleic acid framework is a three-dimensional DNA nanostructure with a tetrahedral shape formed by ingenious DNA sequence design, the application of the principle of complementary pairing, and the automatic hybridization of each strand. Each DNA tetrahedron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/704A61K48/00A61K31/713A61K47/26A61P35/00A61K49/00
CPCA61K31/704A61K31/713A61K47/26A61P35/00A61K49/0032A61K49/0052A61K2300/00
Inventor 宋春元董晨张晶晶汪联辉
Owner NANJING UNIV OF POSTS & TELECOMM
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